Modified bacteriophage

ABSTRACT

Provided is a modified bacteriophage capable of infecting a target bacterium, which bacteriophage includes an α/β small acid-soluble spore protein (SASP) gene encoding a SASP which is toxic to the target bacterium, wherein the SASP gene is under the control of a constitutive promoter which is foreign to the bacteriophage and the SASP gene.

PRIORITY APPLICATION INFORMATION

This application is a divisional of U.S. Application No. 14/190,727,filed Feb. 26, 2014, now U.S. Pat. No. 9,359,596, which is a divisionalof U.S. Application No. 12/672,311, filed Jun. 3, 2010, now U.S. Pat.No. 8,697,049, which is a 371 National Stage of InternationalApplication No. PCT/EP08/60360, filed Aug. 6, 2008, which claimspriority to GB Application No. 0715416.4, filed Aug. 7, 2007, thecontents of all of which are incorporated by reference herein in theirentireties.

The present invention relates to modified bacteriophage and a processfor the production of modified bacteriophage.

SEQUENCE LISTING INFORMATION

The sequence listing file named “43297o2003.txt,” having a size of 2,190bytes and created May 5, 2016, is hereby incorporated by reference inits entirety.

BACKGROUND TO THE INVENTION

Staphylococcus aureus is the most common cause of infections contractedwhilst in hospital (nosocomial infections) (Noskin et al., 2005). Itfrequently causes infections in the lungs, wounds, skin and the bloodand, because of the number of toxins the bacterium can produce, theseinfections may be life threatening.

Almost all strains of S. aureus are now resistant to penicillin owing totheir ability to produce an enzyme (penicillinase) which breaks down thedrug; and 45 years after the introduction of methicillin in 1959, apenicillinase-resistant penicillin, methicillin resistant S. aureus(MRSA) strains are endemic in many hospitals. More recently MRSA strainshave also become a problem in the community. Many MRSA strains are nowresistant to multiple antibiotics.

MRSA levels have risen dramatically in hospitals in both the US and theUK and, in addition, new Community Acquired MRSA (CA-MRSA) strains havespread rapidly across the globe since they were first reported in thelate 1990's. These CA-MRSA strains have proven to be highlytransmissible and often carry a set of genes encoding Panton ValentineLeukocidin which is a toxin that can make these strains highly virulent.There are concerns that these CA-MRSA strains may further add to thedifficulties of controlling MRSA infections in hospitals (Donegan,2006).

In fact, MRSA is now such a serious (and lethal) problem in hospitalsthat significant effort is being put into implementing infection controlmeasures as a way of minimising the spread of MRSA in hospitals and thusreducing the number of infections. In relation to MRSA in particular,infection control measures include, variously, the use of handsanitisers by healthcare workers; screening, isolation and barriernursing of infected and carrier patients; and decontamination ofpatients and healthcare workers who carry MRSA. The carriage of bacteriais defined as the presence of bacteria, usually at a low level, withoutany associated pathology such as inflammation. However, MRSA carriers doconstitute a significant risk for the spread of MRSA to the widerhospital community, and the elimination of MRSA from carriers,particularly on or prior to admission, is a very important part of theinfection control process.

Carriage of S. aureus (and therefore MRSA) occurs in and around thenose, armpits, groin, and perineum as well as in superficial skinlesions. A number of studies report that S. aureus is carried in thenose by 25 to 30% of the general population with MRSA being carried byaround 1%. Amongst hospital patients the carriage rate is significantlyhigher. In the US it has been estimated that 89 million people carry S.aureus in their nose, and 2.3 million of those carry MRSA (Mainous etal., 2006). The intra-nasal elimination of MRSA is therefore fundamentalto controlling the spread of this potentially lethal organism inhospitals.

As an alternative to conventional antibiotics, one family of proteinswhich demonstrate broad spectrum antibacterial activity inside bacteriacomprises the α/β-type small acid-soluble spore proteins (knownhenceforth as SASP). Inside bacteria, SASP bind to the bacterial DNA:visualisation of this process, using cryoelectron microscopy, has shownthat SspC, the most studied SASP, coats the DNA and forms protrudingdomains and modifies the DNA structure (Francesconi et al., 1988;Frenkiel-Krispin et al., 2004) from B-like (pitch 3.4 nm) towards A-like(3.18 nm; A-like DNA has a pitch of 2.8 nm). The protruding SspC motifsinteract with adjacent DNA-SspC filaments packing the filaments into atight assembly of nucleo-protein helices. In this way DNA replication ishalted and, where bound, SASP prevent DNA transcription. SASP bind toDNA in a non-sequence specific manner (Nicholson et al, 1990) so thatmutations in the bacterial DNA do not affect the binding of SASP.

WO02/40678 describes the use as an antimicrobial agent of bacteriophagemodified to incorporate a SASP gene. In order to provide effectiveproduction of the modified bacteriophage in a bacterial host, WO02/40678aims to avoid expression of the SASP gene during proliferation of theproduction host. To this end, the SASP gene was preferably inserted intothe lysis genes of the bacteriophage so as to put the SASP gene underthe control of a lysis gene promoter which is active only at the end ofthe bacteriophage life cycle. It was thought that proliferation of thebacterial production host would otherwise be prevented owing to thepresence of the SASP gene product, particularly if the SASP gene wasunder the control of a constitutive promoter. In a less preferredarrangement, the SASP gene could be located elsewhere on thebacteriophage chromosome and placed under the control of a bacteriophageor bacterial promoter whereby the lytic cycle could be left to run itscourse. In this arrangement, the bacterial promoter would benon-constitutive and could be up-regulated by environmental cues.

SUMMARY OF THE INVENTION

It has now surprisingly been found that effective production ofbacteriophage may be achieved where the bacteriophage has been modifiedto carry a gene encoding a SASP under the control of a promoter which iscontrolled independently of the bacteriophage, and which is constitutivewith no exogenous or in trans regulation necessary or provided, Whenpresent in multiple copies, for example following infection of targetcells, the promoter.drives toxic levels of SASP expression.

Accordingly, in a first aspect, the present invention provides amodified bacteriophage capable of infecting a target bacterium, whichbacteriophage includes an α/β small acid-soluble spore protein (SASP)gene encoding a SASP which is toxic to the target bacterium, wherein theSASP gene is under the control of a constitutive promoter which isforeign to the bacteriophage and the SASP gene.

In a second aspect, there is provided a process for the production of amodified bacteriophage capable of infecting a target bacterium, whichprocess comprises growing a bacterial host comprising genetic materialencoding the bacteriophage in a growth medium; causing the bacteriophageto replicate in the bacterial host; and harvesting the bacteriophage.

Use of a modified bacteriophage in which the SASP gene is under thecontrol of a constitutive promoter has a number of advantages. Controlof expression of the SASP gene is removed from the bacteriophage wherebyproduction of SASP becomes independent of phage gene expression. Thisenables the SASP to be produced even when the bacteriophage cannot carryout its full life cycle; which may happen in the case of super-infection(where the bacteriophage infects a bacterial host already carrying aprophage) and host restriction of the bacteriophage DNA. As describedbelow this strategy thus allows one phage type to inhibit many differentstrains of one bacterial species.

Whilst bacteriophage generally tend to have narrow host ranges, puttinga SASP gene under the control of a constitutive promoter can broaden thehost range of the modified bacteriophage. This is because one of the keyways in which bacteria limit their host range is by degradingbacteriophage DNA on entry into a bacterial cell. Use of bacteriophageto deliver aSASP gene, whose production is independent of thebacteriophage, means that the fate of the bacteriophage DNA may notimpact on the efficacy of the SASP. In this way, the bacteriophage actsas a delivery vector by delivering the gene encoding the SASP to atarget bacterial cell.

Production of a modified bacteriophage according to the inventionrequires a bacterial host which can be lysogenised by the bacteriophage.This lysogen should allow proliferation of the bacteriophage uponinduction, so that an adequate bacteriophage titre may be obtained forharvesting. According to the invention, the SASP do not prevent theproduction of adequate phage titres within the timescale required by amanufacturing process, i.e. prior to host cell death.

A preferred approach according to the present invention is to use aconstitutive promoter to control the SASP gene, such that the promoterdoes not promote the expression of sufficient SASP to kill the hostproduction strain from which the modified bacteriophage is to beharvested. The promoter may be a bacterial promoter, such as from S.aureus. Preferred promoters include the S. aureus promoters pdhA forpyruvate dehydrogenase E1 component alpha subunit, rpsB for the 30Sribosomal protein S2, pgi for glucose-6-phosphate isomerase. Sequenceshaving >90% identity to these sequences may also be used on promotersaccording to the invention. A particularly preferred promoter is thepromoter for the fructose bisphosphate aldolase gene, fbaA, from S.aureus N315 (accession no. BAB43211), or a sequence showing >90%homology to this sequence. An advantage of using the fbaA promoter toexpress the SASP gene is that this promoter expresses constitutively inbacterial cells and does not appear to be regulated by any mechanismwithin S. aureus cells. In addition, a single copy of the fbaA::SASP-Celement does not produce enough SASP-C to be lethal to a host cell,enabling maintenance and production of the PTSA 1.2/A bacteriophage, asdescribed in further detail below. Upon infection of target bacteria,however, multiple copies of the fbaA promoter (from multiple infectionevents or phage replication within the target cell) drives sufficientexpression of SASP-C so as to cause loss of viability of the target.

Thus promoters which are suitable to be used upstream of SASP inbacteriophage constructs, such as the fbaA promoter, have two definingproperties; they are not strong enough to kill the bacteriophage's hostduring growth of the host bacterium; they do not prevent the productionof adequate phage titres within the timescale required by amanufacturing process, i.e. prior to host cell death. However, they aresufficiently strong so as to drive the production of toxic levels ofSASP when present in multiple copies, i.e. following delivery ofmultiple copies of the SASP gene to a targeted cell or due to phagereplication in a targeted cell. Selection of promoters with suchactivities may be made by analysing bacteriophage constructs for thesecharacteristics.

The promoter controlling transcription and therefore expression of theSASP gene is foreign to both the bacteriophage and the SASP gene in thesense that it does not originate from the bacteriophage and is not thenative promoter of the SASP gene. In this way, control of expression ofthe SASP gene is divested from the phage.

The bacterial host can be any host suitable for a given bacteriophage.The host must support the bacteriophage through the proliferation ofmature bacteriophage particles within the host, when induced to do so.WO02/40678 sets out in appendix 4 a list of common pathogens and some oftheir phages. This appendix is reproduced in the present application asappendix 1. Staphylococcus and Clostridium hosts, preferablyStaphylococcus aureus and Clostridium difficile, are particularly usefulhosts. Bacteriophage ø11 is capable of infecting Staphylococcus aureusand is described in further detail below. This bacteriophage may bemodified in accordance with the present invention.

Sequences of α/β-type SASP may be found in appendix 1 of WO02/40678,including SASP-C from Bacillus megaterium which is the preferredα/β-type SASP.

Bacteriophage vectors modified to contain a SASP gene have generallybeen named SASPject vectors. SASPject vector PTSA1.2/A is described infurther detail below for delivery of the SASP gene to S. aureus,including MRSA. Once the SASP gene has been delivered to a targetbacterium, SASP is produced inside those bacteria where it binds tobacterial DNA and changes the conformation of the DNA from B-liketowards A-like. Production of sufficient SASP inside target bacterialcells causes a drop in viability of affected cells; thus toxicity causedby SASP is dose dependent, which in turn is dependent upon promoteractivity and number of promoter::SASP copies present.

In general, the SASP gene and its promoter may be placed anywhere withinthe bacteriophage genome. However, it is preferred for targetingpathogenic bacteria that the modified bacteriophage is non-lytic andthis may be achieved by the removal or inactivation of one or more genesrequired by the phage for lysing infected bacteria, most preferably byinactivating at least one of the lysis genes. In a preferred embodiment,the SASP gene is inserted into one of the lysis genes or the lysis geneis replaced with the toxin gene. The genes for lysing infected bacteriainclude the bacteriophage holin gene and/or an amidase gene. One or moreof these genes may be interrupted or replaced by the SASP gene.Preventing the modified bacteriophage from lysing its target bacterialhost allows continued expression and accumulation of the SASP, possiblybeyond the time at which the bacteriophage would normally cause thebacterial host to lyse.

In a further aspect, the present invention provides a composition forinhibiting or preventing bacterial cell growth, which comprises amodified bacteriophage as defined herein and a carrier therefor. Such acomposition may have a wide range of uses and is therefore to beformulated according to the intended use. The composition may beformulated as a medicament, especially for human treatment and may treatvarious conditions, including bacterial infections. Among thoseinfections treatable according to the present invention are topicalinfections, dental carries, respiratory infections, eye infections andlocalised tissue and organ infections. The carrier may be apharmaceutically-acceptable recipient or diluent. The exact nature andquantities of the components of such compositions may be determinedempirically and will depend in part upon the routes of administration ofthe composition.

Routes of administration to recipients include oral, buccal, sublingual,intranasal, by inhalation, topical (including ophthalmic), intravenous,intra-arterial, intra-muscular, subcutaneous and intra-articular. Forconvenience of use, dosages according to the invention will depend onthe site and type of infection to be treated or prevented. Respiratoryinfections may be treated by inhalation administration and eyeinfections may be treated using eye drops. Oral hygiene productscontaining the modified bacteriophage are also provided; a mouthwash ortoothpaste may be used which contains modified bacteriophage accordingto the invention formulated to eliminate bacteria associated with dentalplaque formation.

A modified bacteriophage according to the invention may be used as abacterial decontaminant, for example in the treatment of surfacebacterial contamination as well as land remediation or water treatment.The bacteriophage may be used in the treatment of medical personneland/or patients as a decontaminating agent, for example in a handwash.Treatment of work surfaces and equipment is also provided, especiallythat used in hospital procedures or in food preparation. One particularembodiment comprises a composition formulated for topical use forpreventing, eliminating or reducing carriage of bacteria andcontamination from one individual to another. This is important to limitthe transmission of microbial infections, particularly in a hospitalenvironment where bacteria resistant to conventional antibiotics areprevalent. For such a use the modified bacteriophage may be contained inTris buffered saline containing CaCl₂ (10 mM) and MgCl₂ (1 mM) or may beformulated within a gel or cream. For multiple use a preservative may beadded. Alternatively the product may be lyophilised and excipients, forexample a sugar such as sucrose may be added.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in further detail, by way ofexample only, with reference to the accompanying drawings and thefollowing example.

BRIEF DESCRIPTION OF THE DRAWINGS:

FIG. 1. Region of S. aureus phage ϕ11, showing the holin and amidasegenes, and the priming sites for amplification of the genes and flankingDNA

FIG. 2. Diagram of pSA1, showing the cloned region and the location ofthe priming sites for inverse PCR of pSA1.

FIG. 3. Diagram of pSA4, showing the cloned promoter-saspC region withthe cadmium resistance (Cd^(R)) gene and the flanking ϕ11 DNA, togetherwith the location of relevant priming sites.

FIG. 4. Arrangement of DNA within the genome of PTL1003, showing thereplacement of the holin gene with foreign genes. The arrangement ofgenes in the wild-type ϕ11 genome is shown for comparison.

FIG. 5. An example of a kill curve showing efficacy of PTSA1.2/A againstan S. aureus strain.

FIG. 6. A kill curve comparing the killing ability of PTSA1.2/A with thesame phage minus the SASP gene.

FIG. 7. A kill curve of PTSA1.2/A infecting an S. aureus strain.

Summary of Construction of a Genetically Altered Bacteriophage CarryingSASP-C Under Control of a Fructose Bisphosphate Aldolase Homologue(fbaA) Promoter

Genes can be removed and added to the phage genome using homologousrecombination. There are several ways in which phages carrying foreigngenes and promoters can be constructed and the following is an exampleof such methods.

For the construction of a ϕ11 derivative it is shown how, using an E.coli/S. aureus shuttle vector, as an example only, the phage holin genehas been replaced with the gene for SASP-C, under the control of a S.aureus fructose bisphosphate promoter homologue (fbaA is used from thispoint on to denote the fructose bisphosphate aldolase promoter). Genesfor resistance to the heavy metal Cadmium (referred to henceforth asCd^(R)) are used as a non-antibiotic resistance marker.

The fbaA-SASP-C and Cd^(R) regions were cloned between two regions ofϕ11 DNA which flank the ϕ11 holin gene. Subsequently, this plasmid wasintroduced into cells and double recombinants were selected for, wherethe holin was replaced with the fbaA-SASP-C and Cd^(R) region.

Experimental Procedures

All PCR reactions were performed using Expand High Fidelity PCR systemand stringent conditions, depending upon the melting temperatures(T_(m)) of the primers, according to the manufacturers instructions.Unless otherwise stated, general molecular biology techniques, such asrestriction enzyme digestion, agarose gel electrophoresis, T4 DNAligase-dependent ligations, competent cell preparation andtransformation were based upon methods described in Sambrook et al.(1989). DNA was purified from enzyme reactions and prepared from cellsusing Qiagen DNA purification kits. S. aureus cells were transformedwith plasmid DNA by electroporation, using methods such as thosedescribed by Schenck and Ladagga (1992).

Primers were obtained from Sigma Genosys. Where primers includerecognition sequences for restriction enzymes, an extra 2-6 nucleotideswas added at the 5′ end to ensure digestion of amplified PCR DNA.

All clonings, unless otherwise stated, are achieved by ligating DNAsovernight with T4 DNA ligase and then transforming them into E. colicloning strains, such as DH5α or XL1-Blue, with isolation on selectivemedium, as described elsewhere (Sambrook et al., 1989)

An E. coli/S. aureus shuttle vector, designated pSM198 was used totransfer to genes between E. coli and S. aureus. Plasmid pSM198 waspreviously produced by combining E. coli cloning vector pUC 18 and thetetracycline resistance and replication regions of S. aureus plasmidpT181. The plasmid carries resistance markers that can be selected forin E. coli and S. aureus. This plasmid retains the pUC18 multiplecloning site (MCS), although not all the sites remain as unique sites.The remaining unique sites in the MCS of pSM198 are: PstI, SalI, BamHI,SacI and EcoRI.

Construction of a Plasmid for Targeted Replacement of the ϕ11 Holin GeneWith fbaA-SASP-C/Cd^(R)

1. Plasmid pSA1, comprising pBluescript SK+ containing a 1.8 kb fragmentof ϕ11 spanning the lytic genes, was constructed as follows. FIG. 1shows the priming sites for the oligonucleotides described below foramplification of regions from the ø11 genome.

PCR amplification of ϕ11 DNA using primers B1001 and B1002, was carriedout and yielded a 1.8 kb fragment which was cleaned and digested withXbaI and PstI. After digestion, the DNA was cleaned and cloned into XbaIand PstI digested pBluescript SK+, yielding pSA1.

Primer B1001 (SEQ ID NO: 1) comprises a 5′ PstI site (underlined)followed by sequence of ϕ11 (Genbank: AF424781) from base 39779 to base39798, (see FIG. 1). Primer B1002 (SEQ ID NO: 2) comprises an XbaI site(underlined) followed by the reverse and complement of sequence of ø11from base 41537 to base 41556 (see FIG. 1).

B1001 (SEQ ID NO: 1) 5′ - AACTGCAGGTGTATTGCAACAGATTGGCTC - 3′ B1002(SEQ ID NO: 2) 5′ - GCTCTAGACTTTGCTCCCTGCGTCGTTG - 3′

2. Inverse PCR was carried out on pSA1 as the template, using primersB1003 (SEQ ID NO: 3) and B1004 (SEQ ID NO: 4) (see FIG. 2).

Primer B1003 comprises a 5′ BamHI site (underlined) followed by thereverse and complement sequence of ϕ11 from base 40454 to base 40469(see FIG. 1). Primer B1004 comprises a 5′ SpeI site (underlined),followed by sequence of ϕ11 from base 40891 to base 40911 (see FIG. 2).

B1003 (SEQ ID NO. 3) 5′ - CGGGATCCGACTAAAAATTAGTCG - 3′ B1004(SEQ ID NO. 4) 5′ - GGACTAGTGAATGAGTATCATCATGGAGG - 3′

This PCR reaction yielded an ˜4.2 kb fragment which constituted: ϕ11left arm, the entire pBluescript SK+ plasmid, and the ϕ11 right arm.This fragment was digested with BamHI and SpeI, cleaned, andsubsequently used as a vector to clone in the following fragment.

3. The cadmium resistance region from pI258 was amplified by PCR usingprimers B1005 and B1006, yielding an ˜2.8 kb fragment. The PCR productwas cleaned and digested with BamHI and XbaI. The digested PCR productwas cleaned and cloned into pSA1 (PCR amplified and digested, above),making pSA2.

Primer B1005 (SEQ ID NO: 5) is complementary to DNA 308 by upstream fromthe ATG for the putative cadmium-responsive regulatory protein gene cadCfrom pI258 (Genbank: J04551), the 3′ end being nearest the ATG (see FIG.3). The 5′ end of the primer carries a non-complementary tail with aBamHI site (underlined) to aid cloning.

Primer B1006 (SEQ ID NO: 6) is complementary to DNA at the 3′ end of thecadA gene for the cadmium resistance protein from plasmid pI258, suchthat the last 3 complementary nucleotides are complementary to the stopcodon TAG of the cadA gene (see FIG. 3). The 5′ end carries anon-complementary XbaI site (underlined) to aid cloning.

B1005 (SEQ ID NO: 5) 5′ - CGATGGATCCTCTCATTTATAAGGTTAAATAATTC - 3′ B1006(SEQ ID NO: 6) 5′ - GCAGACCGCGGCTATTTATCCTTCACTCTCATC - 3′

4. The DNA containing the ϕ11 left and right arms and Cd^(R) were cutout of pSA2 using PstI and SacI, and gel purified away from the vector.This fragment was cloned into shuttle vector pSM198 which was also cutPstI and SacI. Clones were screened for the restriction fragment andcandidates were sent for sequencing. A correct plasmid construct wasidentified and named pSA3. This plasmid was used to clone in thefollowing fragments.

5. PCR amplification of the fbaA promoter using B1007 and B1008 yieldedan approximately 300 bp fragment which was cleaned and subsequentlydigested with NcoI, then re-cleaned.

The fbaA PCR fragment was ligated to the SASP-C coding sequence from B.megaterium. The amplification and preparation of the SASP-C gene isdescribed below.

Primer B1007 (SEQ ID NO: 7) comprises a 5′ sequence tail which includesa BamHI site, followed by the reverse complement of bases 2189404 to2189427 from the S. aureus NCTC 8325 genome (Genbank: CP000253) (seeFIG. 3).

Oligonucleotide B1008 (SEQ ID NO: 8) comprises a sequence tail whichincludes an NcoI site, then the sequence of bases 2189214 to 2189232from the S. aureus NCTC 8325 genome (see FIG. 3). When a PCR product ismade using this primer, the NcoI site incorporated into the primer atthe ATG of the gene results in the change of the base 2 nucleotidesupstream of the ATG from T>C.

B1007 (SEQ ID NO: 7) 5′ - CTACGGATCCTTTATCCTCCAATCTACTTATAAA - 3′ B1008(SEQ ID NO: 8) 5′ - CATGCCATGGAAGTTCCTCCTTGAGTGCT - 3′

6. The SASP-C gene from B. megaterium strain KM (ATCC 13632) wasamplified by PCR with primers B1009 and B1010 and yielded an ˜300 byfragment. The PCR product was cleaned and digested with NcoI. Thedigested PCR product was cleaned and used in a ligation with the fbaAPCR fragment, as described below.

Oligonucleotide B1009 (SEQ ID NO: 9) comprises a 5′ tail containing anNcoI site and is complementary to the first 20 nucleotides of SASP-C(accession no. K01833), starting at the ATG, from B. megaterium strainKM (see FIG. 3). The NcoI site at the beginning of the oligonucleotideincorporates the ATG of the SASP-C gene.

B1009 (SEQ ID NO: 9) 5′ - CGATCCATGGCAAATTATCAAAACGC - 3′

Oligonucleotide B1010 (SEQ ID NO: 10) comprises a BglII site(underlined), and an EcoRI site (double underlined), followed by thereverse complement of DNA starting 59 bases downstream of the stop codonto 74 bases downstream of the stop codon of the SASP-C gene (see FIG.3).

B1010 (SEQ ID NO: 10) 5′ - AGTGAGATCTGAATTCGCTGATTAAAAGAAAC - 3′

7. The fbaA and the SASP-C PCR fragments (both cut NcoI) were ligatedtogether using T4 DNA ligase. The ligated DNAs were used as a templatefor PCR, to amplify the joined fbaA and SASP-C DNAs. PCR was performedusing primers B1007 and B1010. The main PCR product of ˜500 by was gelpurified. The PCR product was digested with BamHI and BglII and cleaned.This fragment was cloned into pSA3 which was prepared as follows. Theplasmid was cut with BamHI, and the ends were dephosphorylated usingcalf intestinal alkaline phosphatase (CIAP). The DNA was cleaned again.

Plasmids were screened so that the end of the SASP-C gene was adjacentto the “left arm” region of ϕ11, and so the start of the fbaA promoterwas adjacent to the cadmium chloride resistance region. The resultingplasmid, carrying fbaA-SASP-C, was named pSA4.

Replacement of the Holin Gene From S. aureus Phage ϕ11 With fbaA-SASP-Cand the Cd^(R) Marker

1. pSA4 was transformed into S. aureus strain PTL47. PTL47 is amonolysogen of ϕ11 in RN4220.

2. Cells which had undergone a double crossover, where the DNA containedbetween the ϕ11 left and right arms of pSA4 have replaced the DNAbetween the ϕ11 left and right arms in the phage genome (ie the holingene) gave rise to colonies with the following phenotype: CdCl₂ (0.1 mM)resistant, tetracycline (5 μg/ml) sensitive. Tetracycline resistance iscarried by the shuttle vector pSM198. Loss of tetracycline resistance isindicative of loss of pSM198. Colonies which had the phentoype: CdCl₂^(R), tetracycline^(S) were screened further by colony PCR.

3. PCR reactions were performed to check that the holin gene was nolonger present, and that the fbaA-SASP-C and the CdCl₂ ^(R) gene werepresent and correctly placed in the ϕ11 prophage genome. PCR fragmentswere sequenced to ensure that the isolate carried the expected sequence,especially in regions: fbaA and SASP-C.

Verified prophage constructs were thus identified and a representativewas picked and named PTL1001.

4. Phage was induced from a culture of strain PTL1001 by heat shock, andthe cells were lysed with lysostaphin (0.25 μg/ml), and then filteredthrough a 0.2 μm filter, yielding a crude cell-free phage lysate.

5. This lysate was used to infect S. aureus strain 8325-4. The infectionmixture was plated onto ϕVPB (vegetable peptone brothcontaining 10 g/lsodium chloride) +CdCl₂ (0.1 mM) agar plates to select for lysogensafter overnight growth at 37 °C.

6. Lysogens were checked by colony PCR as described above. A verifiedlysogen was identified and named PTL1002.

7. PTL1002 was passaged 5 times on ϕVPB agar, picking a single colonyand re-streaking to single colonies at each passage.

8. A single colony was picked and analysed again by PCR and sequencing.The verified isolate was named PTL1003. The phage carried by thislysogen strain is called PTSA1.2/A (see FIG. 4).

SASPject vector PTSA1.2/A has been tested against a panel of S. aureusstrains and clinical isolates, including methicillin sensitive S. aureus(MSSA) and MRSA strains belonging to each of the 5 recognised scc-mectypes. An example of a kill curve showing efficacy of PTSA1.2/A againstan S. aureus strain is given in FIG. 5.

A kill curve comparing the killing ability of PTSA1.2/A versus the samephage minus the SASP gene (phage SAO/A) is given in FIG. 6, and confirmsthat the kill rate is due to presence of the SASP.

A kill curve of PTSA1.2/A infecting an S. aureus strain which is amonolysogen of PTSA1.2/A is given in FIG. 7, and shows thatsuperinfection immunity to the phage does not prevent SASP frominhibiting infected cells.

REFERENCES

-   Donegan, N. 2006. Annual Meeting of the Soc. for Healthcare    Epidemiology of America.-   Francesconi, S. C., MacAlister, T. J., Setlow, B., and    Setlow, P. 1988. Immunoelectron microscopic localization of small,    acid-soluble spore proteins in sporulating cells of Bacillus    subtilis. J. Bacteriol. 170: 5963-5967.-   Frenkiel-Krispin, D., Sack R., Englander, J., E. Shimoni,    Eisenstein, M., Bullitt, Horowitz-Scherer, E. R., Hayes, C. S.,    Setlow, P., Minsky, A., and Wolf, S.G. 2004. Structure of the    DNA-SspC Complex: Implications for DNA Packaging, Protection, and    Repair in Bacterial Spores. J. Bacteriol. 186: 3525-3530.-   Mainous, A. G. III, Hueston, W. J., Everett, C. J., and    Diaz V. A. 2006. Nasal Carriage of Staphylococcus aureus and    Methicillin Resistant S. aureus in the US 2001-2002. Annals of    Family Medicine 4:132-137.-   Nicholson, W. L., Setlow, B., and Setlow, P. 1990. Binding of DNA in    vitro by a small, acid-soluble spore protein from Bacillus subtilis    and the effect of this binding on DNA topology. J. Bacteriol. 172:    6900-6906.-   Noskin, G. A., Rubin, R. J., Schentag, J. J., Kluytmans, J.,    Hedblom, E. C., Smulders, M., Lapetina, E., and Gemmen, E. 2005. The    Burden of Staphylococcus aureus Infections on Hospitals in the    United States: An Analysis of the 2000 and 2001 Nationwide Inpatient    Sample Database. Arch Intern Med 165: 1756-1761-   Sambrook, J., Fritsch, E. F. and Maniatis, T. in Molecular Cloning,    A Laboratory Manual 2nd edn (Cold Spring Harbor Press, New York,    1989).

Schenk, S., and R. A. Laddaga. 1992. Improved method for electroporationof Staphylococcus aureus. FEMS Microbiol. Lett. 73:133-138.

APPENDIX 1

A list of common pathogens and some of their phages. (This list isrepresentative but not exhaustive).

Coliphages:

Bacteriophage lambda Bacteriophage 933W (Escherichia coli O157:H7)Bacteriophage VT2-Sa (E. coli O157:H7) Coliphage 186 Coliphage P1Coliphage P2 Coliphage N15 Bacteriophage T3 Bacteriophage T4Bacteriophage T7 Bacteriophage KU1

Bacteriophages of Salmonella spp

Bacteriophage Felix Bacteriophage P22 Bacteriophage L Bacteriophage 102Bacteriophage  31 Bacteriophage F0 Bacteriophage 14 Bacteriophage 163Bacteriophage 175 Bacteriophage Vir Bacteriophage ViVI Bacteriophage  8Bacteriophage  23 Bacteriophage  25 Bacteriophage  46 Bacteriophage E15Bacteriophage E34 Bacteriophage 9B

Bacteriophages of Shigella dysenteriae

Bacteriophage ϕ80 Bacteriophage P2 Bacteriophage  2 Bacteriophage  37

Bacteriophages of Vibrio cholerae

Bacteriophage fs-2 Bacteriophage 138 Bacteriophage 145 Bacteriophage 149Bacteriophage 163

Bacteriophages of Mycoplasma arthritidis

Bacteriophage MAV1

Bacteriophages of Streptococci

Bacteriophage CP-1 Bacteriophage ϕXz40 Bacteriophage 1A Bacteriophage 1BBacteriophage 12/12 Bacteriophage 113 Bacteriophage 120 Bacteriophage124

Bacteriophages of Pseudomonas aeruginosa

Bacteriophage D3 Bacteriophage ϕCTX Bacteriophage PP7

Bacteriophages of Haemophilus influenzae

Bacteriophage S2 Bacteriophage HP1 Bacteriophage flu Bacteriophage Mu

Bacteriophages of Staphylococcus aureus

Bacteriophage Twort Bacteriophage tIII-29S Bacteriophage ϕPVLBacteriophage ϕPV83 Bacteriophage  ϕ11 Bacteriophage  ϕ12 Bacteriophage ϕ13 Bacteriophage  ϕ42 Bacteriophage ϕ812 Bacteriophage K BacteriophageP3 Bacteriophage P14 Bacteriophage UC18 Bacteriophage  15 Bacteriophage 17 Bacteriophage  29 Bacteriophage  42d Bacteriophage  47 Bacteriophage 52 Bacteriophage  53 Bacteriophage  79 Bacteriophage  80 Bacteriophage 81 Bacteriophage  83 Bacteriophage  85 Bacteriophage  93 Bacteriophage 95 Bacteriophage  187

Bacteriophages of Chlamydia

Bacteriophage ϕCPAR39

Mycobacteriophage

Bacteriophage L5 Bacteriophage LG Bacteriophage D29 Bacteriophage Rv1Bacteriophage Rv2 Bacteriophage DSGA

Bacteriophages of Listeria monocytogenes

Bacteriophage A118 Bacteriophage 243 Bacteriophage A500 BacteriophageA511 Bacteriophage 10 Bacteriophage 2685 Bacteriophage 12029Bacteriophage 52 Bacteriophage 3274

Bacteriophages of Klebsiella pneumoniae

Bacteriophage 60 Bacteriophage 92

Bacteriophages of Yersinia pestis

Bacteriophage R Bacteriophage Y Bacteriophage P1

The invention claimed is:
 1. A method for treating a bacterial infectionin or on a subject comprising administering to said subject atherapeutically effective amount of a modified bacteriophage whichinhibits or prevents growth of a target bacterium, wherein saidbacteriophage includes an α/β small acid-soluble spore protein (SASP)gene encoding a SASP which is toxic to the target bacterium, wherein theSASP gene is under the control of a constitutive bacterial promoterwhich is foreign to the bacteriophage and the SASP gene, which promoterdrives production of toxic levels of SASP when present in multiplecopies in the target bacterium and wherein the bacteriophage contains asingle copy of the SASP gene functionally linked to said constitutivebacterial promoter.
 2. The method of claim 1, wherein said modifiedbacteriophage is applied topically.
 3. The method of claim 1, whichinhibits or prevents bacterial cell growth in said patient.
 4. Themethod of claim 1, wherein said bacteriophage comprises a modifiedStaphylococcus aureus bacteriophage.
 5. The method of claim 4, whereinsaid modified Staphylococcus aureus bacteriophage is a modified ø 11bacteriophage.
 6. The method of claim 1, wherein said bacteriophage isformulated for topical or oral use.
 7. The method of claim 1, whereinsaid bacteriophage is formulated as a gel or cream.
 8. The method ofclaim 1, wherein said constitutive promoter is selected from pyruvatedehydrogenase E1 component alpha subunit (pdhA), ribosomal protein S2(rpsB), glucose-6-phosphate isomerase (pgi), fructose bisphosphatealdolase (fbaA), and sequences having more than 90% identity thereto. 9.The method of claim 8, wherein the bacterial fbaA promoter is fromStaphylococcus aureus.
 10. The method of claim 1, wherein saidbacteriophage is non-lytic.
 11. The method of claim 10, wherein saidSASP gene is inserted into a lysis gene of said bacteriophage.
 12. Themethod of claim 1, wherein said bacteriophage lacks a functional holinggene.
 13. The method of claim 1, wherein said bacteriophage comprises aϕ 11 bacteriophage having a holin gene into which is inserted a geneencoding Bacillus megaterium SASP-C under the control of an fbaAconstitutive promoter.
 14. The method of claim 1, wherein saidbacteriophage comprises a non-antibiotic resistance marker.
 15. Themethod of claim 1, wherein said bacteriophage comprises a cadmiumresistance marker.